The Functional and Antiviral Activity of Interferon Alpha-Inducible IFI6 Against Hepatitis B Virus Replication and Gene Expression

Abstract

Hepatitis B virus is an enveloped DNA virus, that infects more than three hundred and sixty million people worldwide and leads to severe chronic liver diseases. Interferon-alpha inducible protein 6 (IFI6) is an IFN-stimulated gene (ISG) whose expression is highly regulated by the stimulation of type I IFN-alpha that restricts various kinds of virus infections by targeting different stages of the viral life cycle. This study aims to investigate the antiviral activity of IFI6 against HBV replication and gene expression. The IFI6 was highly induced by the stimulation of IFN-α in hepatoma cells. The overexpression of IFI6 inhibited while knockdown of IFI6 elevated replication and gene expression of HBV in HepG2 cells. Further study determined that IFI6 inhibited HBV replication by reducing EnhII/Cp of the HBV without affecting liver enriched transcription factors that have significant importance in regulating HBV enhancer activity. Furthermore, deletion mutation of EnhII/Cp and CHIP analysis revealed 100 bps (1715-1815 nt) putative sites involved in IFI6 mediated inhibition of HBV. Detailed analysis with EMSA demonstrated that 1715-1770 nt of EnhII/Cp was specifically involved in binding with IFI6 and restricted EnhII/Cp promoter activity. Moreover, IFI6 was localized mainly inside the nucleus to involve in the anti-HBV activity of IFI6. In vivo analysis based on the hydrodynamic injection of IFI6 expression plasmid along with HBV revealed significant inhibition of HBV DNA replication and gene expression. Overall, our results suggested a novel mechanism of IFI6 mediated HBV regulation that could develop potential therapeutics for efficient HBV infection treatment.

Keywords: CHIP; EMSA; HBV; IFI6; antiviral activity; interferon-stimulated genes.

Figure 1

Expression level of IFI6 is significantly induced by IFN-α in Hepatocytes. HepG2 and Huh7 cells were seeded in 12 well cell culture plates. After 24 hours, cells were treated with human IFN-α (100 ng/ml). Samples were collected at 0, 4, 8, and 12 hours after IFN-α treatment and total RNA was purified and subjected to qRT-PCR (A, B) and western blot for protein detection (C, D). The anti-IFI6 antibody was used to determine the protein level of IF6. Expression of β-actin was used as a loading control. The data are averages of three independent experiments; the results were measured as a mean ± SD and statistically analyzed. ***P < 0.001, ****P < 0.0001.

Figure 2

IFI6 overexpression inhibits HBV replication and gene expression. HepG2 cells were seeded in 12 well cell culture plates and transfected with indicated amounts of the plasmid. Cells were harvested after 48 h posttransfection (A, B, C, F, G) or 96 hours post-transfection (D, E). (A) The expression of IFI6 mRNA and protein was measured by qRT-PCR and western blot, respectively. The anti-IFI6 and anti-HA-tag antibodies were used to determine the protein level of IF6 and HA-tag, respectively. The β-actin was used as a loading control. (B) HBV RNA transcripts (3.5 kb, 2.4 kb, and 2.1 kb) were determined with northern blot. The amount of 28S and 18S rRNAs was used as a loading control. (C) The level of HBV 3.5 kb RNA was measured with qRT-PCR. The GAPDH level was used as an internal control. HBV core associated (D) and extracellular DNA (E) was determined by qPCR. (F) The level of secreted HBsAg and HBeAg from transfected cell supernatant was measured with ELISA. (G) The cytotoxicity of transfected HepG2 was determined with a CCK8 kit after 48 hours of transfection with the indicated amount of plasmids. The data are averages of three independent experiments; the results were measured as a mean ± SD and were statistically measured. *** P < 0.001, **** P < 0.0001.

Figure 3

Knockdown of IFI6 enhances HBV replication and gene expression. Stable HepG2 cell lines expressing shRNA (control), shIFI6-1, and shIFI6-2 were seeded in 12 well cell culture plate and transfected after 24 hours with pHBV1.3 (800 ng) and β-Gal (200 ng) and cells were harvested 48 hours posttransfection (A, B, C, F, G) or 96 hours posttransfection (D, E). (A) The IFI6 mRNA and the protein expression level were measured by qRT-PCR and western blot, respectively. The anti-IFI6 antibody was used to determine the protein level of IF6. The β-actin was used as a loading control. (B) HBV RNA transcripts were determined with northern blot. The amount of 28S and 18S rRNAs was used as a loading control. (C) The level of HBV 3.5 kb RNA was measured with qRT-PCR. The GAPDH level was used as an internal control. (D) HBV core associated DNA and extracellular DNA (E) was determined with qPCR. (F) The level of secreted HBsAg and HBeAg from transfected cell supernatant was measured with ELISA. (G) The cytotoxicity of the stable knockdown cell line was determined with CCK8 kit after 48 hours of transfection with pHBV (800 ng) and β-Gal (200 ng). The data are averages of three independent experiments; the results were measured as a mean ± SD and were statistically measured. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 4

IFI6 downregulate HBV gene expression by suppressing EnhII/Cp promoter activity. HepG2 cells, along with IFI6 expression plasmid or control plasmid (250 ng) was transiently transfected with four reporter plasmids (200 ng) in 24 well cell culture plates and harvested the cells after 48 hours of transfection and subjected to Dual-Luciferase assay. (A) The activity of four promoters was measured, and the pRL-TK (50 ng) was used as a transfection efficiency control. (B) The activity of the pCMV promoter (200 ng) was measured with a Dual-Luciferase assay. (C) The activity of EnhII/Cp was measured in the indicated dose. (D) The level of different liver enriched transcription factors was measured by qRT-PCR. The + in pHBV1.3 showing 0.4 µg concentration, the + in pSV-β-gal showing 0.2 µg concentration, the + and ++ in pCAGGS and IFI6 indicate 0.2 and 0.4 µg concentration of pCAGGS and IFI6, respectively, and - in pCAGGS and IFI6 panel is showing zero concentration of pCAGGS and IFI6. The data are averages of three independent experiments; the results were measured as a mean ± SD and were statistically measured. *p < 0.05, **p < 0.01, ***P < 0.001, ns, non-significant.

Figure 5

IFI6 suppresses HBV replication by binding to 1715-1770 nt of EnhII/Cp promoter. (A) Schematic diagram of EnhII/Cp promoters and its truncated mutants. (B) The mutants were subcloned into pGL3 basic plasmid and transfected in HepG2 cells. Cells were harvested after 48 h of transfection, and Dual-luciferase activity was measured. (C) HepG2 cells were seeded in a 10 cm dish and transfected with pHBV1.3 (5 µg) with IFI6 (5 µg) or control plasmid (pCAGGS, 5 µg), after 48 hours of transfection, cells were harvested, immunoprecipitate was extracted with overnight incubation of Anti-Polymerase II, mouse IgG, and Anti-HA-Tag antibody to analyze the binding ability of EnhII 1715-1815. The protein-bound EnhII 1715-1815 DNA was extracted, amplified with PCR, and detected with agarose gel electrophoresis. (D) Expression and purification of recombinant GST-IFI6 protein were performed from E.coli. The western blot was performed to measure the size of the GST-tagged IFI6 protein. The arrow indicated the expected band size of protein, and elution 3 was used for subsequent EMSA experiments. (E) EMSA was performed to determine the specific binding of IFI6 with EnhII/Cp 1715-1770 region. Lane 1, GST IFI6 protein alone, Lane 2, Cy5 labeled probe (1715-1770 nt) alone, Lane 3, Cy5 labeled probe (1715-1770 nt) incubated with non-specific competitor probe (Cy5 1415-1470), Lane 4-7, Cy5 labeled probe (1715-1770 nt) incubated with different concentrations of GST IFI6 protein, Lane 8-10, a competition of Cy5 labeled probe (1715-1770 nt) with different concentrations of an unlabeled cold probe (1715-1770 nt). (F) EMSA was used to determine the binding of IFI6 with EnhII/Cp 1760-1815 region. Lane 1, Cy5 labeled probe (1760-1815) alone, Lane 2, GST IFI6 protein alone, Lane 3, Cy5 labeled probe (1760-1815 nt) incubated with non-specific competitor probe (Cy5 1415-1470), Lane 4-7, Cy5 labeled probe (1760-1815 nt) incubated with different concentrations of GST IFI6 protein, Lane 8-10, a competition of Cy5 labeled probe (1760-1815 nt) with different concentrations of an unlabeled cold probe (1760-1815 nt). (G) HepG2 cells were seeded in 12 well cell culture plates and transfected after 24 hours with IFI6 (1 µg) alone or co-transfected with pHBV1.3 (0.5 µg). Cells were harvested after 48 h posttransfection. Nuclear and cytoplasmic protein was extracted and subjected to western blot. The anti-IFI6 and (H) anti-HA-tag antibodies were used to determine the protein level of IF6 and HA-tag respectively from nuclear and cytoplasmic extracted protein. The β-actin was used as a loading control. The data shown in the graph are averages of three independent experiments; the results were measured as a mean ± SD and were statistically measured. * P < 0.05, ** P < 0.01, ns, non-significant.

Figure 6

IFI6 inhibits replication of HBV and gene expression in mice. The IFI6 (20 µg) or its control vector (pCAGGS, 20 µg) and pHBV1.3 (10 µg) plasmid were hydrodynamically injected in eight weeks old male C57BL/6 mice. After 5 days post-injection, mice sera and liver were collected. (A) The protein was extracted from mice liver and expression of IFI6 and HA-tag protein was determined with western blot using anti-IFI6 and anti-HA-tag antibody. The β-actin was used as a loading control. (B) The level of 3.5 kb HBV RNA was measured by qRT-PCR. The level of mice GAPDH was used as an internal control. (C) The HBV DNA was extracted from mice serum, and the level was determined by qPCR. The secreted HBsAg (D) and HBeAg (E) were determined with the ELISA. (F) The immunohistochemical staining of HBcAg from mice liver. The data are averages of three independent experiments; the results were measured as a mean ± SD and were statistically measured. ** P < 0.01, *** P < 0.001.

Figure 7

Mechanism of IFI6 mediated inhibition of HBV replication and gene expression. The type I IFN binds to the cell receptor and induces the JAK-STAT signaling pathway that subsequently induces transcription of ISGs. IFI6 is one of the IFN-stimulated genes that inhibit the transcription of 3.5 kb, 2.4 kb, and 2.1 kb RNA of HBV that subsequently inhibit the translation of different HBV proteins.